Investigating the bifunctional chelate approach for radiopharmaceutical development

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Radiopharmaceuticals are drugs that contain a radioactive atom for the purpose of imaging or therapy of disease. The type of emission determines the applicability for each drug. For instance, alpha and beta particles are used for treatment of disease, while gamma and positron emissions are useful for disease detection. There are three main ways to develop a radiopharmaceutical drug; 1) through direct administration of a radionuclide, 2) incorporating a radiolabel directly into a small molecule, or 3) using the bifunctional chelate approach to indirectly label peptides or antibodies. The final model is the focus of this work. The bifunctional chelate approach involves using a chelate that forms thermodynamically and kinetically inert complexes and also has the ability to couple to the targeting moiety. The work involved with this project is three pronged 1) chelate development for Au-198/199, 2) peptide optimization for Ga-67/68, and 3) comparing in vitro/in vivo behavior of Ga-67 and In-111. In the first project, possible chelates using a bis-thiosemicarbazide chelate backbone were investigated for their ability to form stable complexes with gold. Au-198/199 isotopes offer potential uses for both imaging and therapy. Gold(III) chemistry, however, creates pitfalls to the applicability of these isotopes. Gold(III) has the problem of oxidizing donor ligands, or being reduced to Au(0). The benefit of using bis-thiosemicarbazides based chelates was that there are two non-oxidizable imine nitrogen donor atoms and two stable thiol donor atoms. A variety of gold complexes were made at the macroscopic level, and further studied at the radiotracter level for in vitro and in vivo stability. Results demonstrated that the chelates tested did not provide the necessary stability at the radiotracer level, however, insight into possible reasons for this instability were elucidated. The second project involved optimizing the targeting peptide, bombesin, as an antagonist for in vivo imaging of GRP receptor positive tumors. A series of peptides that differed in their C-terminal ends or 6th position amino acid, were synthesized and coupled to the chelate, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and labeled with Ga-67. Gallium-67 is a useful isotope for single photon emission computed tomography (SPECT) and has been found to form thermodynamically stable complexes with DOTA. This project involved comparing the imaging potential of each peptide analogue by first determining in vitro binding affinity and stability, and then comparing in vivo biodistribution profiles. Results demonstrated that analogues with more electron donating groups on the C-terminus generated greater binding affinity, the addition of (D) amino acids in the 6th position aided in background tissue clearance, and the highest tumor accumulation was demonstrated with the 67Ga-DOTA-8-aminooctanoic acid-[(D)Trp6]BBN(6-13)NHC2H5 conjugate. The final project was to compare the in vitro and in vivo behavior of the same peptide conjugates previously discussed when labeled with Ga-67 or In-111. Gallium and indium both share similar chemistries, however, differences in coordination and size could have dramatic effects on in vitro binding affinity and stability, as well as in vivo clearance rates and pathways. Therefore, the lead compounds from the previous project were further evaluated when labeled with In-111 and compared to the results determined for the Ga-67 conjugates. Results demonstrated that in general Ga-67 conjugates had higher binding affinity, similar stability in human serum, and vast differences in both hydrophobicity and in vivo biodistribution profiles.